Fig. 7: MyCAFs and piCAFs can be induced by TNF-α and TGF-β treatment of ADH1B-NAFs.
From: Fibroblast dynamics in colorectal cancer: stability, plasticity, and novel markers
a Impact of TNF-α (50 ng/ml), TGF-β (5 ng/ml), IL-6 (5 ng/ml) and IFN-γ (5 U/ml) on ADH1B expression in NAFs (n = 4: #3, #8, #12, #14). Gene expression was determined by RT-qPCR and normalized to untreated cells. (Ratio-paired two-tailed t-test, n.s. no significance, *P < 0.05, **P < 0.01). b Western Blot of ADH1B in NAFs (#12) after repeated TNF-α and TGF-β stimulation. GADPH indicates that equal amounts of protein were loaded. c The number of ADH1B positive cells in NAF cultures (n = 3: #3, #12, #14) is reduced upon treatment with TNF-α or TGF-β. d Quantitative evaluation of the results from (c). The total cell count and number of positive cells were determined using ImageJ and averaged among three random areas per sample (unpaired two-tailed t-test, *P < 0.05). e–h Impact of TNF-α and TGF-β on fibroblast subpopulation markers (TGM2, ITGA3, ACTA2, TAGLN, MMP1, MMP3 and CXCL8) in NAFs and CAFs (n = 4 per group: #3, #8, #12, #14). Gene expression was determined by qRT-PCR and normalized to untreated cells. (Ratio-paired two-tailed t-test, n.s. no significance, *P < 0.05, **P < 0.01, ***P < 0.001, n.s. no significance). i Impact of short-time (24 h) or long-term (120 h) stimulation of NAFs by TNF-α or TGF-β on expression of ADH1B, CXCL8 and ACTA2. Cytokines were washed out and gene expression was assessed after 48 h and analyzed by RT-qPCR, normalized to untreated cells (n = 4; #3, #7, #9, #10), (Ratio-paired two-tailed t-test, n.s. no significance, *P < 0.05, **P < 0.01, ***P < 0.001). j–m Impact of DNA-Demethylation on ADH1B, ITGA3 expression in NAFs, ACTA2 and TAGLN expression in CAFs and MMP1, MMP3 and CXCL8 expression in both NAFs and CAFs after 5-Azacitidin treatment. Gene expression was analyzed by RT-qPCR and normalized to DMSO treated cells (n = 5 per group; NAFs: #8, #12, #14, #15, #16; CAFs: #8, #12 #14, #15, #17). (Ratio-paired two-tailed t-test, n.s. no significance, *P < 0.05, **P < 0.01). #, patient numbers as detailed in Supplementary Tables S1, S2.
